The detection of mutations has been an area of great interest in recent years. For example, mutations in certain genes have been associated with a variety of disorders—ranging from blood disorders to cancers. Genetic tests are thus becoming an increasingly important facet of medical care. Consequently, there has been an emphasis on the ability to rapidly and efficiently detect mutations and polymorphisms.
Many electrophoretic techniques have been developed to rapidly screen DNAs for sequence differences by which such mutations can be detected. Denaturing Gradient Get Electrophoresis (DGGE) [Myers, R. M., Maniaris, T. and Lerman, L., Methods in Enzymology, 155, 501–527 (1987)], Constant Denaturant Gel Electrophoresis (CDGE) [Borresen, A. L., et al., Proc. Nat. Acad. Sci. USA, 88, 8405 (1991)], Single Strand Conformation Polymorphism (SSCP) [Orrita, M., et al., Proc. Nat. Acad. Sci. USA, 86, 2766–2770 (1989)], Heteroduplex Analysis (HA) [Nagamine, C. M., et al., Am. J. Hum, Genet., 45,377–399 (19 ?9)] and Protein Truncation Test (PTT) [Roest, P. A. M., et al., Hum. Molec. Genet., 2,1719–1721 (1993)] are frequently used methods. Many labs use combinations of these methods to maximize mutation detection efficiency. All these methods require gel electrophoresis. Methods that do not require gel electrophoresis also exist. For example, selective hybridization on immobilized target sequences allows screening for rare known mutations [Zafiropoulos, A., et al., Biotechniques 223, 1104–1109 (1997)], while mass-spectrometry has been used to detect mutations by analyzing molecular weight of proteins [Lewis, J. K., et al., Biotechniques 24, 102–110 (1998)].
A fundamental problem with currently existing mutation and polymorphism detection methods is that they only screen for mutations on a single gene at a time (i.e. the method is directed to looking at a ‘gene of interest’, that is suspected of having a mutation). Given that the human genome has 50,000–100,000 genes, this is a severe limitation. It is likely that unknown mutations and polymorphisms in several other genes both known and unknown, exist simultaneously with mutations/polymorphisms in the ‘gene of interest’. However, mutations in those other genes would likely not be identified. Therefore a method that can perform ‘mutation/polymorphism scanning’ in for a wide array of genes simultaneously, without the initial need for identifying the gene one is screening would be useful. Gel-electrophoresis-based methods are essentially restricted to examining mutations in a single gene at a time. Attempts have been made to devise non-gel electrophoretic methods to identify mutations, that would not be restricted to a single gene [Cotton et al., Proc. Natl. Acad. Sci. USA vol. 85, pp 4397–4401, (1988)] [Nelson, S. F. et al., Nature Genetics, 4, 11–8, (1993 May)] [Modrich, P., et al., Methods for Mapping Genetic Mutations. U.S. Pat. No. 5,459,039, (1995)]. These methods, however, have had limited success (Nollau P. and Wagener C., Clinical Chemistry 43:1114–1128 (1997)) since they are complicated, typically requiring several enzymatic steps and they result in a large number of false positives, i.e. they frequently score mutations and polymorphisms in normal DNA. It would be desirable to have a method that allows highly sensitive and specific identification and rapid purification of sites that contain mutation/polymorphism over large spans of the genome.
Although DNA arrays and methodologies that can simultaneously scan a large set of DNA fragments for gene expression (e.g. the ‘repertoire’ and amount of genes expressed in normal vs. cancer cells) are known [Wodicka L, Nature Biotechnology 15: 1359–1367 (1997); Lockhart, D J, Nature Biotechnology 14: 1675–1680 (1996); Schena, M., Trends Biotecnnol 16: 301–306, (1998); Yang, T. T., Biotechniques 18: 498–503, (1995)], the ability to scan a large set of random DNA fragments for unknown mutations is a much more demanding process on which the technology is lagging [Ginot F., Human Mutation 10: 1–10 (1997)]. Thus far DNA array-based methods to scan for polymorphisms (SNPs) and mutations has been restricted to specific genes [Lipshutz, R. J., Biotechniques 19: 442–447 (1995); Wang, D. G., Science 280: 1077–1082 (1998)]. Whereas detection of unknown mutations over several genes requires a selectivity and sensitivity not currently achievable by present arrays [Ginot F., Human Mutation 10: 1–10 (1997)]. For example, when it comes to unknown mutation detection, even a single gene with a coding sequence of the size of APC (8.5 kb) is difficult to screen in a single experiment, especially when an excess normal alleles is simultaneously present [Sidransky D., Science 278: 1054–1058 (1997)]. Thus, current arrays do not scan whole genes from the 5′ to the 3′ end, but selectively sample the gene. For example, expression arrays are biased to the 3′ end. It would be desirable to have a methodology that could sample genes over larger portions of a gene and multiple genes. A method that permits identification of mismatches over large spans of the genome would also be desirable.